1. Field of the Invention
The present invention relates to a method and an apparatus for flow type particle image analysis which picks up still images of particles suspended in a sample liquid flow and analyzes the particles picked up in the images, particularly suitable for the particle image analysis of cells or particles in urine or blood.
2. Description of the Related Art
Conventionally, analyzing and classifying corpuscles in blood or cells and particles in urine are done by observing a sample applied on a slide glass with a microscope. In case of urine, since the density of particles is low in urine, a sample is observed after being enriched by a centrifugal separator.
The observation and inspection of particles are automated by using an apparatus in which such a sample as a drop of blood is set at a stage of a microscope after being applied on a slide glass and the stage of the microscope is automatically scanned. Then, the scanning is stopped at the position where the presence of particles is found, and a still image of the particles is picked up. Then the particles in the still image are analyzed and classified by a feature extraction means and a pattern recognition means used in an image processing technique.
The above-mentioned observation and inspection need much time for making a sample, and require difficult work for finding particles by mechanically moving the stage of a microscope to move the particles into an adequate view position of an image pickup region, whereby much time for analysis and a complicate mechanical structure of the apparatus are needed.
As another conventional technique, a flow cytometer method is known. The method optically analyzes a sample which is not applied on a slide glass but flowed in a flow cell where the particles are suspended in the flowing sample.
The method using the flow cytometer observes the strength of fluorescence or scattered light from each particle in the sample and has the ability of analyzing thousands of particles a second.
However, it is difficult to observe feature parameters reflecting a morphological feature of a particle by the method. Therefore, the method cannot classify particles based on the morphological feature as in the conventional microscope method.
As a technique of analyzing and classifying each particle image in a picked up still image of particles in continuous sample flow, techniques described in National Publication for Japan No.500995/1982, and Japanese Patent Application Laid-open No.94156/1988 and No.72554/1992 are known.
The technique described in National Publication for Japan No.500995/1982 devises a method for picking up still images of particles in a sample liquid passing through a special shape of a flow path widening at an image pickup region, by using a flash lamp, and for analyzing the particles in the particle still images.
In the above-mentioned technique, when a particle image enlarged by a microscope is projected on a CCD camera, the flash-lamp pulse light source periodically emits light in synchronizing with the operation of the CCD camera.
The particle still images can be obtained though the particles continuously flow since the emission time of the pulse light source is very short and the CCD camera can pick up 30 pieces of particle still images a second.
In the technique described in Japanese Patent Application Laid-Open No.94156/1988, a particle detection system other than an image pickup system is provided upstream of a particle image pickup region in the sample flow. A particle passing through a flow cell is detected in advance by the particle detection system and a flash lamp emits light at a suitable timing when the particle just passes through a particle image pickup region in the above-mentioned technique.
The technique, without a periodic flash and lamp emission, picks up a particle still image only when a particle is detected, and efficiently accumulates the particle still images. Thus, the technique can avoid picking up a meaningless image having no particle even if the particle density of a sample is low.
Example techniques in which a particle detection part is separately provided in an ordinary flow cytometer and a particle still image is not picked up are described in Japanese Patent Application Laid-Open No.260830/1985, No.231244/1988 and No.245131/1989.
In a flow type particle image analysis apparatus, it is generally necessary to provide a particle detection system in a particle image pickup region or upstream of the region as described in the above-mentioned Japanese Patent Application Laid-Open No.94156/1988 in order to efficiently classify plural kinds of particles in a sample by analyzing still images of particles in a continuous sample flow.
That is, the particle still image is picked up by emitting light from a pulse light source only when a particle passes through a flow cell. This method causes the increase of the sample number to be processed, the reduction of processing time and the improvement of analysis accuracy.
Conventionally, the particle detection is performed by a method of focusing and radiating a laser beam on the sample flow to be analyzed in a flow cell and detecting the light scattered from particles intersecting the laser beam. The scattered light produces a light signal having a strength proportional to an effective scattering cross section of a particle.
The light signal is converted to an electrical signal. The strength of the light signal is affected by the optical refractive index, the optical absorption coefficient, the size and the internal state of a particle, the scattered light detection conditions and so on.
Further, another method of particle detection by forming a particle image on a line image sensor and processing the image is also used for the particle detection.
However, there are the below-mentioned problems in the method of classifying plural kinds of particles in a sample by analyzing the still images of particles in a continuous sample flow.
In the above-mentioned method, the particle images are converted to electrical signals by a TV camera and the arriving time of a particle passing through the particle detection region is random.
Further, one frame of image signals is read out from a CCD TV camera by dividing the frame into two fields of images, namely, an odd field image and an even field image, and taking out the images as electrical signals. The image taking out timing does not depend on the particle arriving detection.
The electric charge accumulated in a photosensitive part of the CCD TV is once transferred to a storage part and the field images are output as the image signals in turn from the storage part.
One pulse signal for reading out a field image is generated according to vertical synchronizing signals. It causes a problem to start picking up the next image, that is, to make a flash lamp emit light for picking up a particle image, when the next particle is detected before two successive pulses for reading out two field images are generated.
If the next image is picked up before two successive pulses for reading out two field images are generated, a double exposure is caused since the transfer of the previous frame image is not completed yet.
In order to resolve the problem, the technique described in Japanese Patent Application Laid-Open No.72544/1992 sets the stroboscope emission timing only during the even field image reading out period and prohibits a flash lamp from emitting light during the odd field image reading out period. That is, the particle detection is permitted only during the even field image reading out period and suppressed during the odd field image reading out period in which the light emission of a flash lamp is prohibited.
Therefore, even if a particle exists in the image pickup region during the odd field image reading out period, a particle image is not picked up since the light emission of a flash lamp is not generated. Then, although the method does not cause a problem if the particle density of an analyzed sample is high and the light emission of a flash lamp is generated in every even field image reading out period, it is probable that a particle image cannot be picked up in spite of the chance for picking up a particle image if the particle density of an analyzed sample is low, since the light emission of a flash lamp is not generated on the chance.
Even if the particle detection system is provided and the number of particles to be processed in a unit time is reduced, it does not sufficiently improve the accuracy of analyzing the sample of low particle density, and also causes the problem of low processing efficiency.
Usually, a field image reading out pulse signal is generated in tens of us after a vertical synchronizing signal, and its width is narrow unlike the vertical synchronizing signal.
Generally, the light emission of a flash lamp is prohibited during the predetermined period before the field image reading out pulse signal is generated in order to prevent multiple exposure of a CCD TV camera. The multiple exposure cannot be sufficiently prevented by prohibiting the light emission of a flash lamp only during the above-mentioned period.
Then, another period of prohibiting the light emission following the above-mentioned period is necessary to transfer the electric charge of one field image accumulated in the photo-electric element of a CCD TV camera to a vertical register in synchronism with the vertical synchronizing signal.
The "another period" is a period in which the accumulated electric charge is transferred to the vertical register and the photo-electric element returns to the state capable of accumulating electric charge.
The light emission during the above-mentioned period results in the superposition of a new image on the image to be transferred and in failing to accumulate the electric charge of a normal image signal, whereby a problem that the light emission of a flash lamp must be prohibited during the above-mentioned period is caused.